Influence of toxic metals on activity of acid and
alkaline phosphatase enzymes in metal-contaminated landfill
soils.

Abstract:

A study was conducted to determine the effect of toxic metals on
soil acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1)
enzyme activities in landfill soils. The enzyme activities were
consistently higher in the landfill soils than in an uncontaminated
alluvial soil. The landfill soils contained higher concentrations of
metals (iron, manganese, cadmium, lead, zinc, copper) than did the
alluvial soil. Enzyme activities were negatively correlated with the
metals, with inhibition increasing with the bioavailability of the
metals. It is suggested that the metals affected enzyme activities by
behaving synergistically or additively with each other. Although the
landfill soils had higher enzyme activities than the alluvial soil due
to higher organic matter concentrations, the ratios of enzyme
activity/organic carbon indicated that inhibition of enzyme synthesis
and stability had occurred due to metal stress.

The preservation of soil quality is essential for sustainable
agriculture. Organic matter is a vital attribute of soil quality. The
scarcity of traditional manure and abundance of organic wastes, such as
sewage sludge and municipal solid-waste compost, have led to the
recycling of these organic wastes for use in crop production. According
to Stratton et al. (1995), these wastes contain metals, which have the
potential for contaminating the food chain. Several metals also have a
detrimental effect on soil microbiological and biochemical parameters
(Giller et al. 1998). The need for field studies lies in the fact that
laboratory experiments, where usually large doses of metals are added
only once, are very different from the field situation. In the field,
pollution usually occurs for many years, with the soil microorganisms
being challenged with a mixture of metals. Metals have very long
half-lives, and once they enter the soil, they persist over a long
period (McGrath 1987). Fresh additions of metals in the laboratory,
therefore, do not model their long-term effects on soil microbial
biomass and activity. The assay of soil enzymes can be a sensitive
indicator for estimating the relative pollution effects of metals and
other industrial pollutants in soil. The strong inhibition of a variety
of enzyme activities in metal-polluted soils has been reported (Tyler
1974; Juma and Tabatabai 1977). In recent years, soil biochemical
parameters have been seen to be early and sensitive indicators of soil
stress and can be used to predict long-term trends in soil quality
(Saviozzi et al. 2002).

Phosphorus availability may be the most limiting factor for plant
growth. Much of the plant's demand for phosphorus is met by cycling
of phosphorus in organic matter. Since plants can utilise only inorganic
phosphorus (Stevenson 1986), organic phosphorus compounds, which mostly
originate from plant roots, fungi, and soil microorganisms, must first
be hydrolysed by phosphatase enzymes to the inorganic form (Dick et al.
1983). According to Nannipieri et al. (1990), phosphatase measured in
soils reflects the activity of enzymes bound to soil colloids and humic
substances, free phosphatases in the soil solution, and phosphatases
associated with living and dead plant or microbial cells. Phosphatase
activities can be a good indicator of the organic-phosphorus
mineralisation potential and biological activity of soils (Dick et al.
1983).

Soil enzymes can consist of intracellular and extracellular
components. Extracellular enzymes like acid and alkaline phosphatase can
be stabilised in a 3-dimensional network of organo-mineral complexes and
maintain their activities (Burns 1982). The activities of these
extracellular enzymes are, however, affected by soil constituents
(Gianfreda and Bollag 1994), pH, and trace metals (Eivazi and Tabatabai
1990), because these factors modify the conformation of the enzymes
(Geiger et al. 1998). Furthermore, Eivazi and Tabatabai (1990) assumed
that metal ions might inhibit enzyme reactions: (i) by complexing with
the substrate, (ii) by combining with the protein-active group of the
enzymes, or (iii) by reacting with the enzyme-substrate complex.
Therefore, it is imperative to consider soil properties along with the
metals in order to examine the influence of the metals on soil acid and
alkaline phosphatase activities.

The disposal of huge amounts of solid wastes, both domestic and
industrial, in any metropolitan city is a global problem. In Kolkata,
about 3500 tonnes of solid wastes are generated daily and dumped in the
low-lying areas situated at the eastern outskirt of the city, locally
known as dhapa. Cultivation of vegetables with municipal solid waste and
sewage water, either full or in part, at dhapa has continued for about a
century. Olaniya et al. (1995) reported that the soils of the dhapa
landfill were more heavily contaminated with metals than normal arable
land and are subjected to severe anthropogenic stress and are
potentially hazardous in nature.

A short-term study with municipal solid-waste compost revealed that
there was no detrimental influence on soil microbial and biochemical
parameters (Bhattacharyya et al. 2001), but studies on long-term effects
are scarce. In the absence of any data on phosphatase activity in
landfill soils, and dhapa soil in particular, the objective of this work
was to ascertain whether metals in the landfill soil are a threat to the
soil environment for sustainable agriculture. Both acid and alkaline
phosphatase activities were determined and comparisons made with
activities in uncontaminated arable land. The importance of studying
these activities lies in their vital role in the phosphorus cycle and
their sensitivity towards heavy metals (Juma and Tabatabai 1978).

Materials and methods

Soils were collected during the early summer season from a normal
alluvial soil tract (S1) covered with grassland and 5 different zones
(S2, S3, S4, S5, S6) of dhapa landfill area that were originally on the
same alluvial soil type. A description of the landfill and agricultural
land is presented in Table 1. The sampling locations were about 700 m
apart. Five surface soil samples (0-15 cm) from each zone (total 30
samples) were collected with a spade and brought to the laboratory in
sealed polythene bags and passed through a 2-mm sieve. The sampling was
designed to be representative of the different sites.

Physico-chemical analyses were carried out with air-dried soil
samples. The pH was determined in a 1:2.5 soil:water suspension, whereas
sand, silt, and clay percentages were measured by the International
Pipette Method (Piper 1966). Organic carbon (C) was determined as in
Nelson and Sommers (1982) and total nitrogen according to the method of
Sankaram (1966). Available phosphorus (Olsen P) was estimated according
to Black (1965). Total, DTPA-extractable, and water-soluble metals were
determined by the methods of Page et al. (1982), Lindsay and Norvell
(1978), and Ma and Uren (1998), respectively. Acid and alkaline
phosphatase activities of the soils were estimated by the methods of
Tabatabai and Bremner (1969) with field-moist samples. The data given in
the table were mean of triplicate laboratory analyses.

Statistical analyses were performed with the help of the IRRISTAT
statistical package (version 3/93) developed by IRRI, Philippines.

Results and discussion

Physico-chemical analysis

The landfill soils contained a much higher percentage of sand
(44-69%), but a lesser percentage of silt and clay, than the alluvial
soil; soil texture varied from sandy clay loam to clay (Table 2). All
soil samples were slightly alkaline. Organic C varied significantly
among the soils and was 4-7 times higher in the landfill soils than in
the alluvial soil. The difference in organic C among the landfill soils
seemed to be related to their history of land filling with the
organic-rich solid waste. The landfill soils also contained
significantly higher amounts of total N and available phosphorus than
the alluvial soil. The landfill soils had higher CEC than alluvial soil.
The status of metals in the soils is given in Table 3. The landfill
soils had significantly higher concentrations of total, DTPA-extractable
and water-soluble iron, manganese, cadmium, lead, zinc, and copper than
the alluvial soil. There were also significant variations in the metal
concentrations among the landfill soils.

Enzyme activities

Plant roots are major producers of acid phosphatase enzymes (Speir
and Cowling 1991), but they do not produce alkaline phosphatase.
Alkaline phosphatase enzymes originate from soil bacteria, fungi, and
fauna (Tarafdar and Claassen 1988). Soil microbes can produce and
release large amounts of extracellular phosphatase due to their large
combined biomass, high metabolic activity, and short life cycles (Speir
and Ross 1978).

In high-pH soils, alkaline phosphatase generally exceeds acid
phosphatase activity (Eivazi and Tabatabai 1977). In our alkaline soils,
alkaline phosphatase activity was 1.3-2.1 times higher than acid
phosphatase activity. The higher enzyme activities recorded in the
landfill soils (Table 4) than the alluvial soil could be attributed to
their higher organic C concentrations (Table 2). The negative influence
of metals on enzyme activities (Tyler 1981) was masked in the landfill
soils because of their high organic matter content and, probably, high
microbial biomass, and hence high enzyme activities. The variation in
enzyme activities in the landfill soils is related to the interplay of
physico-chemical properties as well as the bioavailability of the
metals. Possibly, the metal form(s) in the landfill soils were not
sufficiently bioavailable to adversely affect the microbial populations.
Chander et al. (2001) emphasised that apart from metals, other factors
such as soil organic matter also regulate the amount of microbial
biomass, and hence enzyme activity, in soil.

Expressing enzyme activities on an organic C basis can give an
improved understanding of metal stress (Tscherko and Kandeller 1997).
Dick (1994) also had suggested relating soil enzyme activities to soil
organic C. On this basis, activities were higher in the alluvial soil
(S1) than in the landfill soils (Table 4). Our study therefore suggested
that, although the landfill soils had higher enzyme activities than the
alluvial soil due to their high organic matter concentrations, there was
actually an inhibition of enzyme synthesis and stability due to metal
stress.

Relationships between enzyme activities and other soil properties

Regression analysis of the mean values of data from each of the 5
landfill sites showed that the coefficient of determination ([R.sup.2])
of the relationships between acid phosphatase activity and organic C or
pH, as separate variables, were 0.41 and 0.94, respectively. When
activity values were regressed on both organic C and pH, the [R.sup.2]
increased significantly to 0.98. The [R.sup.2] values for alkaline
phosphatase activity with organic C, pH, and organic C combined with pH
were 0.45, 0.90, and 0.98, respectively. Frankenberger and Dick (1983)
and Gianfreda and Bollag (1994) also found the same relationships. These
positive relationships indicate that the enzyme activities may be
primarily associated with the soil organic fraction and stabilised as
enzyme-organic matter complexes.

Both acid and alkaline phosphatase activities were positively
correlated with Olsen-extractable P (r = 0.85, 0.95 respectively; P <
0.05). Cbunderova and Zubets (1969) (cited in Speir and Ross 1978)
reported increased phosphatase activity with increasing P fertilization
until the soluble P content of the soil reached 200 mg/kg soil.
Phosphatase activity declined and disappeared completely at critical
soluble P contents of 600-800 mg P/kg soil. In our study,
Olsen-extractable P ranged from 18 to 85 mg/kg soil, which was low
enough not to retard activity. The high correlation coefficients (r =
0.98; P < 0.05) between acid and alkaline phosphatase activities in
our soils were probably due to the fact that both enzymes were
correlated with organic C concentrations. This result was consistent
with the findings of Frankenberger and Dick (1983).

Influence of metals on the enzyme activities

Metal ions are assumed to inactivate enzymes by reacting with
sulfhydril groups, a reaction analogous to the formation of a metal
sulfide. Sulfhydril groups in enzymes serve as integral parts of the
catalytically active sites or as groups involved in maintaining the
correct structural relationship of the enzyme protein (Juma and
Tabatabai 1977). Metals can reduce enzyme activity by interacting with
the enzyme substrate complex, by denaturing the enzyme protein, or
interacting with the protein active groups (Nannipieri 1995). They can
also affect the synthesis of the enzymes by the microbial cells. Metals
in soils are present in various forms due to interactions with various
soil components; therefore, the total concentrations in soils cannot
provide a precise index for evaluating their influence on soil
microorganisms and enzyme activities (Kunito et al. 2001). Water, and
DTPA- and EDTA-type extractants are more popular reagents to extract
bioavailable metals (Kabata-Pendias and Pendias 1992).

The enzyme activities of the landfill soils were negatively
correlated with the different fractions of all the metals, being lowest
for total concentrations and highest for water-soluble concentrations
(Table 5). Hattori (1992) also found that water-soluble forms of metals
were more toxic than insoluble forms. Among the metals studied,
DTPA-extractable copper and cadmium were most toxic, as reflected by
their comparatively high negative correlation values with both enzyme
activities. It has been generally recognised that copper and cadmium are
more toxic than the other metals (Hiroki 1992). However, it is difficult
to determine whether copper and cadmium strongly affected the soil
enzyme activities individually, because there were highly positive
correlations (P < 0.05) among all the metals studied (r values ranged
from 0.88 to 0.99; mean 0.97) and the changes in the concentrations of
the metals in the field were similar to each other (Table 3). It is thus
suggested that the metals affected enzyme activities in combination with
each other. Beckett and Davis (1978) previously found that when metals
were present in a mixture they behaved synergistically or additively.

Conclusion

The landfill soils were heavily contaminated with metals. Although
the landfill soils had higher enzyme activities than the alluvial soil
because of their high organic matter content, inhibition of enzyme
synthesis and stability due to metal stress is, nevertheless, indicated.

References

Beckett PHT, Davis RD (1978) The additivity of the toxic effects of
Cu, Ni and Zn in young barley. New Phytoloist 81, 155-173.

Tarafdar JC, Claassen N (1988) Organic phosphorous compounds as a
phosphorous source for higher plants through the activity of
phosphatases produced by plant roots and microorganisms. Biology and
Fertility of Soils 5, 308-312.